JSRV-env慢病毒过表达载体构建及其对NIH3T3细胞增殖的影响

Construction of JSRV-env lentiviral vector and its influence on proliferation of NIH3T3 cells

为研究绵羊肺腺瘤病毒(Jaagsiekte sheep retrovirus,JSRV)囊膜蛋白(Envelope,env)对NIH3T3细胞(小鼠胚胎成纤维细胞)增殖的影响,构建pCMV-dR8.91-JSRV-env慢病毒过表达载体并感染NIH3T3细胞。将JSRV-env基因连接红色荧光慢病毒报告载体pCMV-dR8.91,基因测序正确后将载体命名为pCMV-dR8.91-JSRV-env。将pCMV-dR8.91-JSRV-env重组质粒和包装辅助质粒共转染HEK 293T细胞包装产生JSRV-env重组慢病毒。将重组慢病毒悬液利用超速离心沉淀法浓缩,real-time PCR测定病毒滴度。浓缩病毒转导NIH3T3细胞72h后,噻唑蓝(MTT)检测细胞增殖情况。结果显示:env同源重组入pCMV-dR8.91载体,测序结果与预期序列一致;pCMV-dR8.91-JSRV-env与包装辅助质粒共转染HEK 293T细胞72h,real-time PCR验证JSRV-env过表达极显著(P<0.01),JSRV-env重组慢病毒平均滴度为2.99×10^8 IU/mL;MTT分析显示10μg JSR-env慢病毒感染NIH3T3细胞72h显著促进NIH3T3细胞增殖(P<0.05)。综上,成功构建了JSRV-env慢病毒过表达载体,证实JSRV-env能够促进NIH3T3细胞增殖。

To study the effect of the envelope(env)of Jaagsiekte sheep retrovirus(JSRV)on the proliferation of NIH3T3 cells(mouse embryo fibroblast cells),pCMV-dR8.91-JSRV-env lentiviral overexpression vector was constructed and infected with NIH3T3 cells.The JSRV-env gene was ligated into the red fluorescent lentiviral reporter vector pCMV-dR8.91,and the gene was correctly sequenced and named pCMV-dR8.91-JSRV-env.The pCMV-dR8.91-JSRV-env recombinant plasmid and packaging helper plasmid were co-transfected into HEK 293 Tcell packaging to produce recombinant JSRV-env lentivirus.The recombinant lentivirus suspension was concentrated by ultracentrifugation,and the virus titer was determined by real-time PCR.After the concentrated virus transduced NIH3T3 cells for 72 h,thiazole blue(MTT)was used to detect cell proliferation.The results showed that:env gene was completely and correctly recombined into the pCMV-dR8.91 vector by sequencing;pCMV-dR8.91-JSRV-env was co-transfected with packaging helper plasmids into 293 Tcells for 72 h.Real-time PCR verified that JSRV-env was very significantly overexpressed(P<0.01)and the average titer of JSRV-envlentivirus was 2.99×10^8 IU/mL;MTT analysis showed that infection of NIH3T3 cells by 10μg JSRV-env lentivirus for 72 hsignificantly promoted the proliferation of NIH3T3 cells(P<0.05).In conclusion,the JSRV-env lentiviral overexpression vector was successfully constructed,and JSRV-env can promote the proliferation of NIH3T3 cells.